TY - JOUR
T1 - Production of the stable human histamine H 1 receptor in Pichia pastoris for structural determination
AU - Shiroishi, Mitsunori
AU - Kobayashi, Takuya
AU - Ogasawara, Satoshi
AU - Tsujimoto, Hirokazu
AU - Ikeda-Suno, Chiyo
AU - Iwata, So
AU - Shimamura, Tatsuro
N1 - Funding Information:
This work was supported by the ERATO Human Receptor Crystallography Project from the Japan Science and Technology Agency and by the Targeted Proteins Research Program of MEXT (S.I.), Japan . The work was also partly funded by Grant-in-Aid for challenging Exploratory Research (T.S.) and Grant-in-Aid for Scientific Research (B) (T.S.), Grant-in-Aid for Young Scientists (B) (M.S.) the Mochida Memorial Foundation for Medical and Pharmaceutical Research (T.S., T.K.), Takeda Scientific Foundation (M.S.) and the Sumitomo Foundation (T.K.).
PY - 2011/12
Y1 - 2011/12
N2 - G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H 1 receptor (H 1R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H 1R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.
AB - G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H 1 receptor (H 1R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H 1R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.
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U2 - 10.1016/j.ymeth.2011.08.015
DO - 10.1016/j.ymeth.2011.08.015
M3 - Article
C2 - 21903167
AN - SCOPUS:84855989800
SN - 1046-2023
VL - 55
SP - 281
EP - 286
JO - Methods
JF - Methods
IS - 4
ER -